Recombinant agents affecting thrombosis

ABSTRACT

Analogs of Factor Xa (Factor Xai) which are inactive as proteases in the prothrombinase reaction are useful in treatment of diseases characterized by thrombosis. These antithrombotic agents can be conveniently prepared using recombinant techniques.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.07/808,329, filed as PCT/US91/06337 Sep. 4, 1991, now abandoned, whichis a continuation-in-part of U.S. Ser. No. 578,646, filed Sep. 4, 1990,now U.S. Pat. No. 5,278,144.

TECHNICAL FIELD

The invention relates to peptide drugs for prevention or treatment ofthrombosis. More specifically, the invention concerns analogs of FactorXa which lack protease activity and which interfere with the ability ofendogenous Factor Xa to effect the conversion of prothrombin tothrombin.

BACKGROUND ART

Thrombin is a multifunctional protease that regulates several keybiological processes. For example, thrombin is among the most potent ofthe known platelet activators. In addition, thrombin is essential forthe cleavage of fibrinogen to fibrin to initiate clot formation. Thesetwo elements are involved in normal hemostasis but in atheroscleroticarteries can initiate the formation of a thrombus, a major factor inpathogenesis of vasoocclusive conditions such as myocardial infarction,unstable angina, nonhemorrhagic stroke and reocclusion of coronaryarteries after angioplasty or thrombolytic therapy. Thrombin is also apotent inducer of smooth cell proliferation and may therefore beinvolved in a variety of proliferative responses such as restenosisafter angioplasty and graft-induced atherosclerosis. In addition,thrombin is chemotactic for leukocytes and may therefore play a role ininflammation. (Hoover, R. J., et al. Cell (1978) 14:423; Etingin, O. R.,et al., Cell (1990) 61:657.) These observations indicate that inhibitionof thrombin formation or inhibition of thrombin itself may be effectivein preventing or treating thrombosis, limiting restenosis andcontrolling inflammation.

The formation of thrombin is the result of the proteolytic cleavage ofits precursor prothrombin at the Arg-Thr linkage at positions 271-272and the Arg-Ile linkage at positions 320-321. This activation iscatalyzed by the prothrombinase complex, which is assembled on themembrane surfaces of platelets, monocytes, and endothelial cells. Thecomplex consists of Factor Xa (a serine protease), Factor Va (acofactor), calcium ions and the acidic phospholipid surface. Factor Xais the activated form of its precursor, Factor X, which is secreted bythe liver as a 58 kd precursor and is converted to the active form,Factor Xa, in both the extrinsic and intrinsic blood coagulationpathways. It is known that the circulating levels of Factor X, and ofthe precursor of Factor Va, Factor V, are on the order of 10⁻⁷ M. Therehas been no determination of the levels of the corresponding activeFactors Va and Xa.

The complete amino acid sequences of human Factor X and Factor Xa areknown. FIG. 1 shows the complete sequence of the precursor form ofFactor X as described by Davie, E. W., in Hemostasis and Thrombosis,Second Edition, R. W. Coleman et al. eds. (1987) p. 250. Factor X is amember of the calcium ion binding, gamma carboxyglutamyl(Gla)-containing, vitamin K dependent, blood coagulation glycoproteinfamily, which also includes Factors VII and IX, prothrombin, protein Cand protein S (Furie, B., et al., Cell (1988) 53:505).

As shown in FIG. 1, the mature Factor X protein is preceded by a40-residue pre-pro leader sequence which is removed during intracellularprocessing and secretion. The mature Factor X precursor of Factor Xa isthen cleaved to the two-chain form by deletion of the three amino acidsRKR shown between the light chain C-terminus and activationpeptide/heavy chain N-terminus. Finally, the two chain Factor X isconverted to Factor Xa by deletion of the "activation peptide" sequenceshown at the upper right-hand portion of the figure (numbered 1-52),generating a light chain shown as residues 1-139, and a heavy chainshown as residues 1-254. These are linked through a single disulfidebond between position 128 of the light chain and position 108 of theheavy chain. As further indicated in the figure, the light chaincontains the Gla domain and a growth factor domain; the proteaseactivity resides in the heavy chain and involves the histidine atposition 42, the aspartic acid at position 88, and a serine at position185, circled in the figure.

There are two known pathways for the activation of the two-chain FactorX in vivo. Activation must occur before the protease is incorporatedinto the prothrombinase complex (Steinberg, M., et al., in Hemostasisand Thrombosis, Coleman, R. W., et al. eds. (1987) J. B. Lippencott,Philadelphia, Pa., p. 112). In the intrinsic pathway, Factor X iscleaved to release the 52-amino acid activation peptide by the "tenase"complex which consists of Factor IXa, Factor VIII and calcium ionsassembled on cell surfaces. In the extrinsic pathway, the cleavage iscatalyzed by Factor VIIa which is bound to a tissue factor on membranes.Of interest herein, however, is the ability to convert Factor X toFactor Xa by in vitro cleavage using a protease such as that containedin Russell's viper venom. This protease is described by DiScipio, R. G.,et al., Biochemistry (1977) 6:5253.

Returning to the function of Factor Xa per se, the activity of Factor Xain effecting the conversion of prothrombin to thrombin is dependent onits inclusion in the prothrombinase complex. The formation of theprothrombinase complex (which is 278,000 fold faster in effecting theconversion of prothrombin to thrombin than Factor Xa in soluble form)has been studied (Nesheim, H. E., et al., J Biol Chem (1979) 254:10952).These studies have utilized the active site-specific inhibitor, dansylglutamyl glycyl arginyl (DEGR) chloromethyl ketone, which covalentlyattaches a fluorescent reporter group into Factor Xa. Factor Xa treatedwith this inhibitor lacks protease activity, but is incorporated intothe prothrombinase complex with an identical stoichiometry to that ofFactor Xa and has a dissociation constant of 2.7×10⁻⁶ M (Nesheim, M. E.,J Biol Chem (1981) 256:6537-6540; Skogen, W. F., et al., J Biol Chem(1984) 256:2306-2310; Krishnaswamy, S., et al., J Biol Chem (1988)263:3823-3824; Husten, E. J., et al., J Biol Chem (1987)262:12953-12961).

Known methods to inhibit the formation of the prothrombinase complexinclude treatment with heparin and heparinlike compounds. This resultsin inhibition of the formation of the complex by antithrombin III inassociation with the heparin. Other novel forms of Factor Xa inhibitioninclude lipoprotein-associated coagulation inhibitor (LACI) (Girard, T.J., et al., Nature (1989) 338:518; Girard, T. J., et al., Science (1990)248:1421), leech-derived antistatin (Donwiddie, C., et al., J Biol Chem(1989) 264:16694), and tick-derived TAP (Waxman, L., et al., Science(1990) 248:593). Alternatively, agents which inhibit the vitaminK-dependent Gla conversion enzyme, such as coumarin, have been used.None of these approaches have proved satisfactory due to lack ofspecificity, the large dosage required, toxic side effects, and the longdelay in effectiveness.

Accordingly, the invention offers an alternative approach of enhancedspecificity and longer duration of action in inhibiting the formation ofan active prothrombinase complex.

DISCLOSURE OF THE INVENTION

The invention provides effective therapeutic agents for the preventionand treatment of thrombus formation and other pathological processes inthe vasculature induced by thrombin such as restenosis and inflammation.This is highly significant as thrombus formation is the leading cause ofdeath in Western societies, and restenosis is an expanding problem withincreased use of angioplasty and other invasive procedures. Thetherapeutic materials of the invention are inactive forms of humanFactor Xa which are nevertheless capable of incorporation into theprothrombinase complex, thus preventing the formation of activeprothrombinase complex from endogenous Factor Xa. These pharmaceuticalsare especially useful in acute settings to prevent thrombosis. Thisincludes preventing thrombus formation in the coronary arteries ofpatients with rest angina, preventing rethrombosis after thrombolysis,and prevention of thrombosis during complicated angioplasties. Thesepharmaceuticals will also be useful in preventing smooth muscle cellproliferation following angioplasty or other vascular invasiveprocedures. The inventive therapeutics offer considerable advantage overthe now standard treatment which involves heparin (Hanson, R. S., etal., Proc Natl Acad Sci (1988) 85:3184). The compounds of the inventionare double- or single-chain polypeptides which are capable ofparticipation in the prothrombinase complex, but which result in aninactive complex.

In one aspect, the invention is directed to a two-chain polypeptide,designated Factor Xai, which is capable of forming the prothrombinasecomplex, but which results in a complex that lacks proteolytic activity.This two-chain polypeptide may be formed from one of two types of novelprecursors. One type, designated herein Factor Xi, has substantially theamino acid sequence of Factor X, but is modified as described herein soas to result in an inactive two-chain polypeptide, Factor Xai, whencleaved by normal coagulation processing proteases or by in vitrotreatment with Factor X activator from viper venom. The other type,designated herein Factor X'i, is a truncated form of single chain FactorX wherein the proteolytic cleavage site (or portion or extensionthereof) at the C-terminus of the light chain, shown as RKR in FIG. 1,is ligated directly (with the optional addition of one or several aminoresidues) to the N-terminus of the activated form of the heavy chain asshown in one embodiment in FIG. 3. Upon cleavage, Factor X'i alsoresults in the two-chain Factor Xai of the invention which results in aprothrombinase complex lacking proteolytic activity. Of course, theactive cofactor, Factor Xa, could also be generated by using theanalogous precursors of the Factor X' type illustrated in FIG. 2.

Thus, in other aspects, the invention is directed to the Factor Xaitwo-chain prothrombinase complex, and to the novel precursors of theFactor Xai therapeutic proteins, to the DNA sequences encoding them, andto recombinant materials and methods generally which permit theirproduction.

Other aspects of the invention include pharmaceutical compositions ofthe therapeutically useful Factor Xai proteins and to methods to preventor treat thrombosis or other pathological events initiated by thrombinusing these compositions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the structure of human Factor X and its relevant cleavagesites as described in the prior art.

FIG. 2 shows the structure of one embodiment of a single-chain Factor X'which is a precursor to yield a two-chain cleavage product that willparticipate in prothrombinase complex formation. The form shown in thisfigure will produce a two-chain peptide which retains proteolyticactivity in the complex; a modified form, as described below, iscatalytically inactive.

FIG. 3 shows one embodiment of Factor X'i.

FIG. 4 shows the cDNA sequence encoding Factor X.

FIGS. 5(a) and (b) are a Western blot of recombinantly produced,potentially active Factor X and Factor Xa.

FIG. 6 is a Western blot of recombinantly produced, inactivated forms ofFactor X and Factor Xa.

FIGS. 7a-7d consist of a series of Lineweaver-Burk plots showing theenzymatic activity of native and recombinantly produced Factor Xconverted to activated form.

FIGS. 8a-8d show a comparison of prothrombinase complex activity ofvarious Factor X forms.

FIG. 9 shows the result of a two-stage prothrombin clotting assay forvarious forms of Factor X.

FIG. 10 shows inhibition of prothrombinase complex formation by inactiveforms of Factor X.

MODES OF CARRYING OUT THE INVENTION

In general, one aspect of the invention encompasses the therapeuticallyuseful two-chain polypeptide, designated Factor Xai herein, and thesingle-chain precursors of this two-chain protein. These peptides areabout 80% homologous, preferably about 90% homologous to the amino acidsequences shown at positions 1-139 (light chain) and 1-254 (heavy chain)in FIG. 1. It should be noted that in FIG. 1, the pre-pro leadersequence is numbered -40 through -1, prior to the numbering beginning atthe N-terminus of the light chain. The light chain is numbered 1-139.The intervening tripeptide RKR, which, in mature Factor X, is deleted,is not numbered. The activation peptide beginning subsequent to thisintervening tripeptide is numbered 1-52; the isoleucine referred tohereinbelow as "position 53" of the activation peptide is, in fact, thefirst amino acid of the heavy chain in the activated form. This restartsthe numbering shown in the figure, and the heavy chain is numbered1-254.

The embodiments of the two-chain peptide, Factor Xai, are effective informing the prothrombinase complex, as determined by their ability toinhibit (or compete with) the formation of the native prothrombinasecomplex involving Factor Xa. Their ability to inhibit prothrombinasecomplex formation can be determined conveniently by the method ofKrishnaswamy, S., J Biol Chem (1988) 263:3823-3834, cited above.However, when incorporated into the prothrombinase complex, the complexfails to show its proteolytic activity, as determined by the method ofvan Dieijen, G., et al., J Biol Chem (1981) 256:3433 or of Skogen, W.F., et al., J Biol Chem (1984) 256:2306. These Factor Xai proteins mayor may not be immunoreactive with antibodies raised against nativeFactor Xa or against Factor X, including commercially availableantibodies specific for human Factor X. The Factor Xai proteins areantithrombotic materials.

The invention is also directed to precursors of the foregoing inactivecompetitors with Factor Xa. One group of these precursors are novelmodified forms of Factor X designated Factor X', wherein one or more ofthe residues at position 42, 88 or 185 of the heavy chain are convertedto alternate amino acid residues, thus inactivating the proteolyticproperties of the peptide. The modified forms of Factor X contain at aminimum the light chain sequence and the heavy chain sequence to whichis attached the activation peptide. The intervening tripeptide (betweenthe C-terminus of the light chain the N-terminus of the activationpeptide) and the pre-pro leader sequence may or may not be present.Thus, the Factor X' may either be a single-chain protein (when thetripeptide is included) or a two-chain precursor of Factor Xa (when thetripeptide has been deleted).

Preferably, the alteration at the residues of the protease active siteis either a deletion, or a conversion to a conservative, substitutedamino acid so as to maintain the three-dimensional conformation of thetwo-chain protein. By "conservative" is meant a substitution whichmaintains the correct conformation, rather than a substitution whichmaintains the correct activity. Thus, the histidine residue at position42 is preferably replaced by phenylalanine; the aspartic acid atposition 88 is preferably replaced by asparagine or glutamine, and theserine residue at position 185 is preferably replaced by alanine orglycine.

Another group of precursors to the antithrombotic dimeric peptides ofthe invention is designated Factor X'i. In Factor X'i precursors, atleast a substantial portion of the activation peptide, preferably theentire activation peptide, is deleted. The precursors to the two-chainform of Factor X'i, however, must retain a proteolytic cleavage sitebetween the light and heavy chains. Therefore, amino acids subject toendogenous proteolysis are conveniently included in a single-chainprecursor form which extends the carboxy terminus of the light chain byvirtue of the cleavage site to the N-terminus of the heavy chain. Atypical embodiment of the single-chain precursor (including the pre-proleader) to the two-chain Factor X'i, which-will now automatically beactivated by virtue of the absence of the activation peptide sequence(thus, becoming a Factor Xai) is shown in FIG. 3. In this embodiment,the hexapeptide sequence RKRRKR connects the C-terminus of the lightchain directly to the isoleucine residue at the N-terminus of the heavychain. Cleavage of this single-chain Factor X'i results in X'ai. Inconstructing such modified X'i type precursors, a hydrophobic amino acidmust be retained at the N-terminus of the heavy chain (nativelyisoleucine). See, for example, Dayhoff, M. O., "Atlas of ProteinSequence and Structure" (1972) 5:89-99 (Biomed. Res. Foundation, Wash.D.C.) and Greer, J., J. Molec. Biol. (1981) 153:1043-1053.

It is evident that the single-chain Factor X' precursors are also noveland, when cleaved by proteolysis, yield Factor Xa, the normalenzymatically-active form of the dimeric protein. This correspondingconstruction is shown in FIG. 2.

Thus, when the single-chain precursors of either Factor Xa or Factor Xaiare produced recombinantly in suitable host cells, the endogenousenzymes of the host cell may (1) cleave the single-chain precursorFactor X or X' to a two-chain form and, in the case of single-chainFactor X or X', further activate the factor by cleavage of theactivation peptide; in the case of Factor X' single-chain precursors,there is no activation peptide present, so the single-chain precursorwill automatically be activated when cleaved into a dimeric peptide. ForFactor X precursors, the double-chain form containing the activationpeptide may also be cleaved in vitro using a suitable protease, such asthe Factor X activator of Russell's viper venom. Either Factor Xa orFactor Xai will be obtained depending on whether the active site hasbeen inactivated by alteration at the appropriate codons as furtherdescribed hereinbelow.

To summarize the terminology used in this application, the followingglossary may be useful:

"Factor X" refers to the native or recombinantly produced single- ortwo-chain Factor X sequence, essentially as shown in FIG. 1, containingat a minimum the heavy chain to which is attached the activationpeptide, at its N-terminus, and the light chain. These may or may not belinked through a cleavage sequence as indicated in the figure. "rX"refers specifically to the recombinantly produced form of this factor.

"Factor Xi" refers to the recombinantly produced form of Factor X whichlacks proteolytic activity by virtue of the modification of the activesite as described above. The designation "rXi" is also used for thisprotein.

"Factor Xa" refers to native or recombinantly produced, enzymaticallyactive dimer containing light and heavy chain only. The activationpeptide is not present in this complex. "rXa" refers specifically tothis complex when produced recombinantly.

"Factor Xai" refers to the modified form of Factor Xa which is activatedin the sense that it combines to form the prothrombinase complex, butwhich has no serine protease activity by virtue of the modification ofits active site. As this protein is produced only by recombinantmethods, "rXai" is also used to designate this complex.

"Factor X'" refers to a modified, single-chain form of Factor X whichincludes only the light chain, heavy chain, and an intermediate,specific proteolytic cleavage site, such as that shown in FIG. 2. Thissingle-chain precursor may also contain the pre-pro sequence. As it is aresult only of recombinant production, it is also designated "rX'." Whencleaved by a protease so as to become activated, the products areindistinguishable from Factor Xa (or rXa) and, accordingly, thisterminology is again used.

Similarly, "Factor X'i" refers to a modified form of Factor X' which hasbeen inactivated at its catalytic site as described above. One form isshown in FIG. 3. Upon conversion to the two-chain form, as theactivation peptide is not present in the precursor, the products areindistinguishable from Factor Xai or rXai.

Preparation of the Inventive Peptides

The genomic organization and coding sequence, for human Factor X areknown and the cDNA has been retrieved and sequenced (Leytus, S. P., etal., Proc Natl Acad Sci USA (1984) 81:3699; Kaul, R. K., et al., Gene(1986) 41:311-314). The complete cDNA sequence (except for nucleotides1251-1300 which can be deduced from the Kaul et al. reference) is shownin FIG. 4.

Full-length Factor X cDNA inserts are subcloned into M13mp18 or M13mp19vectors for site-directed mutagenesis. (The correct sequence encodingFactor X is verified by dideoxy sequencing.) Standard modificationtechniques are now readily available in the art, and thus the sequenceencoding Factor X is modified to obtain the DNA-encoding Factor X',Factor Xi, and Factor X'i.

The modified coding sequences for Factor X', Factor Xi and Factor X'iare then ligated into suitable expression vectors for recombinantproduction of the polypeptides. In the expression vectors, the preproleader sequence is preferably retained for expression in compatible hostcells such as mammalian hosts. If bacterial or yeast expression isdesired, it may be desirable to substitute a compatible leader sequence,such as the penicillinase sequence in bacteria, or the alpha-factorsequence in yeast. Alternatively, an ATG start codon may be directlyplaced before amino acid 1 of the light chain-encoding sequence toproduce an intracellular protein.

The choice of host and expression control system is governed by thenature of the desired result. If endogenous activation by proteolyticcleavage is desired, mammalian systems may be preferable. However,production in microorganisms which provide simplicity of culturing isnot precluded, provided an in vitro system for carboxylation to producethe required carboxy glutamyl residue is employed, or the microorganismor other host natively lacking this posttranslational processing systemis transformed to provide it. A wide variety of expression systems forrecombinant DNA sequences is known in the art.

The modified DNA encoding Factor X', Factor Xi or Factor X'i ispreferably provided with linkers for ligation into cloning andexpression vectors. Techniques for preparation of such vectors are wellunderstood in the art. The DNA encoding the desired Factor X', Factor Xior Factor X'i is ligated in operable linkage with control sequences,including promoters, upstream enhancers, termination sequences, and soforth, depending on the nature of the intended recombinant host cells.Technology is currently available for expression of heterologous genesin a variety of hosts, including procaryotic hosts and variouseucaryotes, including yeasts, mammalian or avian or insect cells, andplant cells. The choice of control sequences and markers in theexpression vectors is selected appropriately to these hosts.

For example, in procaryotic hosts, various promoters, includinginducible promoters such as the trp promoter and lambda phage P_(L)promoter can be employed. Hybrid promoters such as the tac promoter,which contains the trp polymerase binding region in combination with thelac operator, can be used. Suitable markers are generally those relatedto antibiotic resistance. On the other hand, in mammalian cell cultures,commonly used promoters are virally derived, such as the early and lateSV40 promoters and adenovirus promoters. Mammalian regulatablepromoters, such as the metallothionein-II promoter may also be used. Themetallothionein-II promoter is regulated by glucocorticoids or heavymetals. These promoter systems are compatible with typical mammalianhosts, the most commonly used of which is Chinese hamster ovary (CHO)cells.

Another commonly employed system is the baculovirus expression systemcompatible with insect cells. Plant cells, used in conjunction with, forexample, the nopaline synthetase promoter, and yeast cells, used inconjunction with promoters associated with enzymes important in theglycolytic pathway, can also be employed. A number of suitableexpression systems can be found in appropriate chapters in "CurrentProtocols in Molecular Biology, " Ausubel, F. M., et al , eds.,published by Wiley Interscience, latest edition.

Administration and Use

The Factor Xai peptides of the invention are prothrombinase inhibitorsand are thus useful in procedures complicated by thrombosis and inconditions whose pathogenesis involves thrombin generation. Theseconditions include those involving arterial thrombosis, such as unstable(i.e., rest) angina and abrupt vessel closure during vascularinterventions including coronary and peripheral angioplasty andatherectomy, and during and after vascular bypass procedures (peripheraland coronary), reocclusion after thrombolytic therapy for myocardialinfarction, thrombotic stroke (stroke in evolution), and thrombosis dueto vasculitis (Kawasaki's disease). Also included are conditionsinvolving venous thrombosis, such as deep venous thrombosis of the lowerextremities, pulmonary embolism, renal vein, hepatic vein, inferior venacava thrombosis, and cavernous sinus thrombosis. Other target conditionsare those involving diffuse activation of the coagulation system, suchas sepsis with disseminated intravascular coagulation, disseminatedintravascular coagulation in other settings, thrombotic thrombocytopenicpurpura, and rare conditions of unknown etiology (Lupus anticoagulant).

The Factor Xai of the invention is also useful as an anticoagulant andanti-inflammatory for cardiopulmonary bypass, in harvesting organs, inpreparation of blood products or samples and in transport andimplantation of organs and associated treatment of the recipient. TheFactor Xai, in a slow release form, is especially useful in indwellingintravascular devices (i.v.s, catheters, grafts, patches).

Thrombosis also plays a role in restenosis following vascularinterventions such as angioplasty, atherectomy, or endarterectomy bydirectly or indirectly causing smooth muscle cell proliferation, and theFactor Xai of the invention is also useful in treating this condition.

Adult respiratory distress syndrome (ARDS) is thought to be an"endotoxin" disease in which a prothrombotic endothelium is likely toexist, with inflammatory and proliferative components; Factor Xai isalso useful in treatment of ARDS.

The therapeutic Factor Xai peptides of the invention are formulated foradministration using excipients conventional for administration ofproteins, typically by injection, as set forth, for example, inRemington's Pharmaceutical Sciences, Mack Publishing Company, latestedition, Easton, Pa. For the antithrombosis effect, the Factor Xaiproteins are administered systemically, preferably by injection, andpreferably by intravenous injection. Dosage levels depend on a number offactors, including the condition of the subject and the specific FactorXai embodiment chosen. However, suitable dosage ranges are on the orderof 1-50 mg per patient per continuous injected dose. For injection, theprotein is dissolved or suspended in liquid medium, for example, Hank'ssolution, Ringer's solution, dextrose solution, and various buffers.Additional excipients such as stabilizers can also be employed.

Besides injection, the peptides of the invention can be administeredsystemically, via suppository, oral administration, transmucosaladministration, including intranasal sprays, and by slow releaseformulations. Additional formulation techniques include encapsulationformulations, such as liposomes.

In addition to utility as a therapeutic, the Factor Xai can be used toraise polyclonal antisera or to produce cells which can be fused toimmortalizing partners to obtain sources of monoclonal antibodiesspecific for this peptide. These antibodies are useful as passivetherapeutics or as diagnostic tools.

The following examples are intended to illustrate, but not limit theinvention.

EXAMPLE 1 Construction of DNA Encoding Catalytically Inactive Forms ofRecombinant Human Factor X (rXi)

A full length cDNA clone for human Factor X was obtained from Dr. W. R.Church, University of Vermont (FIG. 4). This cDNA encodes the amino acidsequence of FIG. 1 or an allelic variant. This human Factor X cDNA wascloned into EcoRI site of vector pBSII (Stratagene) to obtain pBSX. TheHindIII-XbaI fragment of pBSX comprising the entire Factor X codingregion was subcloned into the Hind III-XbaI site of vector M13mp19(Mp19X). Oligonucleotide site-directed mutagenesis was then performed asdescribed by Kunkel, T. A., et al., Methods in Enzymol (1987) 54:367.

The following forms were produced:

The oligomer TGC CGA GGG GAC GCC GGG GGC CCG CAC was used to convertserine (S₁₈₅) at position 185aa on the Factor X heavy chain to alanine(A₁₈₅) to obtain rXiA₁₈₅.

The oligomer ACC TAT GAC TTC AAC ATC GCC GTG CTC was used to convertaspartic acid (D₈₈) at position 88aa on the Factor X heavy chain toasparagine (N₈₈) to obtain the gene encoding rXiN₈₈.

Both oligomers were used to obtain the gene encoding rXiN₈₈ A₁₈₅. (SeeFIG. 1 for location of these sites). Verification ofoligonucleotide-directed mutagenesis was accomplished by dideoxysequencing.

EXAMPLE 2 Construction of DNA Encoding the Truncated Precursor of HumanFactor Xa (rX')

The cDNA of human Factor X (Mp19X) was converted to encode varioustruncated forms of human Factor Xa, collectively designated rX', bydeletion of the activation peptide, by oligonucleotide site directedmutagenesis (Kunkel, T. A., et al., Methods in Enzymol (1987) 154:367).The following oligonucleotides employed with the corresponding aminoacid changes are as follows:

rX'ΔO: ACC CTG GAA CGC AGG AAG AGG ATC GTG GGA GGC CAG GAA TGC, whichaligned arginine (R₁₄₂) following the C-terminus of the Factor X lightchain with isoleucine (I₅₃) 53aa of the Factor X activation peptide (1aaof the heavy chain);

rX'Δ1: ACC CTG GAA CGC AGG AAG AGG AGA ATC GTG GGA GGC CAG GAA TGC,which aligned this R₁₄₂ with arginine (R₅₂) of the Factor X activationpeptide;

rX'Δ2: ACC CTG GAA CGC AGG AAG AGG CGG AAA AGA ATC GTG GGA GGC CAG GAATGC, which extended R₁₄₂ following the Factor X light chain by two aminoacids arginine (R₁₄₃) and lysine (K₁₄₄) and aligned this terminus withR₅₂ of the Factor X activation peptide (FIG. 2);

rX'Δ3: ACC CTG GAA CGC AGG AAG AGG CCT AGG CCA TCT CGG AAA CGC AGG ATCGTG GGA GGC CAG GAA TGC, which extended R₁₄₂ following the Factor Xlight chain by seven amino acids, Proline (P₁₄₃) Arginine (R₁₄₄) Proline(P₁₄₅) Serine (S₁₄₆) Arginine (R₁₄₇) Lysine (K₁₄₈) Arginine (R₁₄₉) asdescribed in Ehrlich, H. J., et al., J Biol Chem (1989) 264:14298, andaligned this terminus with R₅₂ of the Factor X activation peptides.

Verification of the oligonucleotide directed mutagenesis wasaccomplished by dideoxy sequencing.

As will be further described below, the precursor derived from rX'Δ2 wascleaved endogenously when recombinantly produced in CHO cells to obtaindirectly the activated form rXa. The precursor derived from rX'Δ0 wasnot cleaved endogenously in CHO cells when produced recombinantly. Theprecursor derived from rX'Δ1 or from rX'Δ3 was cleaved incompletely. Thedimeric peptides derived from rX'Δ0, rX'Δ1 and rX'Δ3 were not activeenzymatically.

EXAMPLE 3 Construction of DNA Encoding Catalytically Inactive TruncatedPrecursor (rX'i)

cDNA Factor X' constructs described in Example 2 were converted toencode the catalytically inactive forms of X' (rX'i) by oligonucleotidesite-directed mutagenesis as described in Example 1. These constructsincluded rX'i(Δ2)N₈₈ and rX'i(Δ2)N₈₈ A₁₈₅, as shown in FIG. 3.

EXAMPLE 4 Expression of the Genes Encoding Precursor (rX and rX')

The expression vector pRC/CMV (Invitrogen) was modified by replacing theCMV promoter with the SRα promoter (Takabe, Y., et al., Molec Cell Biol(1988) 8:466). The ClaI-XbaI fragment, filled in by Klenow polymerase atthe ClaI site which contained the SRα promoter was isolated from theexpression vector pBJ1 (Lin, A., et al., Science (1990) 249:677 andavailable from M. Davis, Stanford University) and subcloned into theNruI-XbaI site of pRC/CMV creating expression vector pBN. The StuIfragment of pBN, comprising the SRα promoter, bovine growth hormonepolyadenylation site and M13 origin or replication was subcloned intothe StuI site of pSV2DHFR generating expression vector pBD. The Mp19SmaI-EcoRV fragments of the precursor DNAs described in examples weresubcloned into the Klenow polymerase-filled-in XbaI site of pBN and pBD.

The resulting expression vectors were transfected into CHO bylipofection (BRL). Selection for transfected clones was by either 1mg/ml G418 Neomycin (Gibco) or 25 ng/ml Methotrexate (Sigma). Singleclones were isolated by cloning cylinders, expanded and expressionlevels were determined on 24 hour serum free medium by a standard solidphase antibody capture assay (ELISA) as described by Harlow, E., andLane, D., in Antibodies (1988), Cold Spring Harbor Laboratory, New York.The ELISA utilized a primary antibody of rabbit polyclonal antihumanFactor X (STAGO, American Diagnostics Inc.) and a rabbit-specificsecondary antibody of peroxidase conjugated goat IgG.

Clones from constructs pBNX, pBNX'ΔO, pBNX'Δ1, pBNX'Δ2, and pBNX'Δ3 wereexpanded to confluency in T-75 tissue culture flasks in RPMI mediumsupplemented with 10% fetal bovine serum, Penicillin, Streptomycin,Glutamine and 10 μg/ml vitamin K, washed four times with serum freemedium and incubated overnight with serum free medium.

Post-incubation the medium was harvested, centrifuged at 3000 rpm and 2ml was precipitated with 10% Trichloracetic acid (TCA). The TCA pelletwas washed three times with 100% Acetone, resuspended to 0.05 mlSDS-PAGE sample buffer or 0.05 m SDS-PAGE sample buffer with 1M βMercaptoethanol. Duplicate 10 μl aliquots were electrophoresed on 12%SDS polyacrylamide gels and transferred to Immobilon filters(Millipore). Western blot analysis was performed with the primary humanFactor X polyclonal rabbit sera (STAGO, American Diagnostics, Inc.) at a1/4000 dilution in 1% nonfat dry milk, 0.1% NP40, 10MM Tris-HCl pH 7.5,150 mm NaCl. The secondary antibody was ¹²⁵ I labeled Fab donkeyantirabbit IgG (Amersham). Autoradiography was overnight at -70° C. withan intensifier screen.

The pattern of antibody reactivity showed that the expected productswere produced. All five products, i.e., those derived from rX, RX'Δ0,rX'Δ1, rX'Δ2, and rX'Δ3 were positive in the above ELISA based on rabbitpolyvalent human Factor X antisera. ELISAs were also performed withrespect to mouse monoclonal antibodies Mab323, Mab743 and Mab325. Mab323is specifically reactive with the activation peptide. Mab743 is reactivewith either the activated or inactivated form of human. Factor X. Mab325is calcium ion dependent and directed to the light chain; this antibodyreacts with the gamma-carboxylated region.

Supernatants from cultures containing any of the five constructs gavepositive ELISAs with Mab743 and Mab325; thus, posttranslational GLAprocessing is indicated in all cases. All of the rX' mutants failed toreact with Mab323 confirming the absence of the activation peptide.

FIG. 5 shows Western blot analysis using polyclonal rabbit antisera ofproducts derived from rX, rX'Δ0, rX'Δ1, rX'Δ2, rX'Δ3 and CHO controlmedium. Rabbit polyclonal antisera to X was not efficient in localizingthe fully processed heavy chain of human Factor Xa; hence, in all casesthe position expected to be occupied by the activated heavy chain doesnot appear. FIG. 5a shows reduced and FIG. 5b nonreduced forms of theserecombinant proteins. Lane 1, 0.7 μg native human Factor X (Dr. C.Esmon, OMRF, University of Oklahoma); Lane 2, rX; Lane 3, rX'Δ0; Lane 4,rX'Δ1; Lane 5, rX'Δ2; Lane 6, rX'Δ3; Lane 7, CHO control medium.

FIG. 5a shows that the recombinant products of rX and rX'Δ2 are dimericproteins which are separable under reducing conditions. The products ofexpression of rX'Δ0, rX'Δ1 and rX'Δ3 apparently are largely single-chainproducts. It appeared that the unprocessed Factor X' single chainscomigrated anomalously with the heavy chain as shown in lanes 3-7,apparently due to the degree of proteolytic processing of the novelcleavage sites.

The failure of these X' precursor proteins to be processed properly wasconsistent with the results of a coagulation assay, described in Example8, which demonstrated that Factor Xa, RVV-activated Factor X orrecombinant Factor X and X'Δ2 were comparably active, while theremaining X' secreted products were dramatically less efficient, by atleast 5 orders of magnitude. The data with respect to enzyme activityare shown in Table 1:

                  TABLE 1    ______________________________________                           Catalytic             RVV           Effi-      Coagu-    Factor X Activation    ciency (%) lation    ______________________________________    X        +             100        +    Xa       -             851        +    rX       +             29.6       +    X'Δ0             -             5.2 × 10.sup.-4                                      -    X'Δ1             -             12.6 × 10.sup.-4                                      -    X'Δ2             -             269        +    X'Δ3             -             69.5 × 10.sup.-4                                      -    CHO      -             0          -    ______________________________________

The column of Table 1 labeled "catalytic efficiency" shows theamidolytic substrate activities of the various factors, activated withRVV if necessary. The catalytic efficiencies shown are the ratio ofkcat/Km and were normalized to the results for plasma Factor X. As shownin the table, both recombinant Factor X and X'Δ2 were active in Factor Xdependent 2PT clotting assays, while the enzymatic activities of theother recombinant proteins were 4 orders of magnitude lower.

From FIG. 5b, it is apparent that the expression products of theX'-encoding gene are of lower molecular weight than rX or native FactorX.

EXAMPLE 5 Purification of rX and X'Δ2

Both recombinant Factor X and X'Δ2 were purified to homogeneity asfollows: After growth to confluency, CHO cells transfected with pBNX orpBNX'Δ2 were washed 4-5 times with serum-free media. The cells were thencultured for consecutive 24 hr periods at 37° C. in serum-free mediasupplemented with 4μg/ml vitamin K₃.

Harvested media were centrifuged at 15,000× g for 20 min followed byfiltration of the supernatant through a 0.2 μm filter. To the media wasadded Tris HCl, pH 7.5 to 20 mM, NaEDTA to 10 mM, and the resultant waschromatographed on Q-Sepharose Fast Flow (Pharmacia). Allchromatographic steps were performed at 4° C. The columns were washedextensively with 20 mM Tris, pH 7.5, 10 mM EDTA, 0.15M NaCl , and theproteins were eluted with 20 mM Tris, pH 7.5, 0.5M NaCl, 5 mM CaCl₂.Peak fractions were pooled and either stored frozen at -20° C. orapplied directly to an anti-factor X monoclonal antibody affinity columnas described by Church, W. R., et al., Throm Res (1985) 38:417-424. Theantibody used for isolation (aHFX-1d, Mab B12-A3) is specific for humanfactor X, not influenced by Ca²⁺, and binds both factors X and Xa(unpublished data). Factor rX' was purified further on abenzamidine-Sepharose column (Pierce) as described by Krishnaswamy, etal., J Biol Chem (1987) 262:3291-3299. The concentrations of theproteins were determined by quantitative ELISA, colorimetric proteinassay (Harlow, E., et al., "Antibodies, A Laboratory Manual" (1988),Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y.), and byabsorbance measurement at 280 nm using extinction coefficient 11.6 andmolecular weights of 58,900 for factor X, 46,000 for X'Δ2.

The purified factors, when subjected to SDS-PAGE under reducingconditions and silver stained showed that the recombinantly producedFactor X was separated into 3 bands representing the full-lengthprecursor (75 kD), the heavy chain containing the activation peptide (45kD) and the light chain (22 kD). When amino terminal sequence analysiswas performed following electrotransfer to nylon filters, the lightchain was shown to be heterogeneous with 27% initiating at Val₃₇ and 73%initiating at Ala₄₁ ; the 75 kD species was also heterogeneous with 41%initiating at Val₃₇ and 59% initiating at Ala₄₁.

EXAMPLE 6 Expression of Genes Encoding Inactivated Recombinant HumanFactor X (rXi and rX'i)

The X' form chosen for conversion to the inactive form was the rX'Δ2form shown in FIG. 4. pBN-derived cell lines for rX, rX'(Δ2), rXiN₈₈A₁₈₅, rXiA₁₈₅, rX'i(Δ2)N₈₈ A₁₈₅ and rX'i(Δ2)N₈₈ were grown to confluencyin 800 cm² roller bottles as described in Example 4, washed four timeswith serum free medium and incubated overnight with 50 ml serum-freemedium. The medium was replenished and harvested daily.

Consecutive harvests were pooled, centrifuged at 3000 rpm and passeddirectly through a Factor X-specific monoclonal antibody (Mab) affinitycolumn (Mab717) supplied by Dr. C. Esmon (OMRF, University of Oklahoma).The bound "Factor X" was eluted from the Mab717 column with 80% ethyleneglycol, dialyzed against 10 mM Tris HCl, pH 7.5, 150 mM NaCl andconcentrated on a Centricon 10 filtration unit (Amicon). "Factor X"protein concentrations were determined by ELISA as described in Example4 utilizing serial dilution with comparison to a standard preparation ofhuman Factor X (Haematologic Technologies, Inc., C. Esmon, OMRF,University of Oklahoma).

The purified proteins were characterized by Western blot analysis asoutlined in Example 4. FIG. 6 shows a Western blot of theseβ-mercaptoethanol-reduced, Mab717 purified recombinant human Factor Xanalogs. Lane 1, 0.1 μg human X (Haematologic Technologies, Inc.); Lane2, 0.1 μg human Xa (Haematologic Technologies, Inc.); Lane 3, 0.1 μg rX;Lane 4, 0.16 μg rX'Δ2; Lane 5, 0.13 μg rXiN₈₈ A₁₈₅ ; Lane 6, 0.15 μgrXiA₁₈₅ ; Lane 7, 0.187 μg rX'i(Δ2)N₈₈, Lane 8, 0.05 μg rX'i(Δ2)N₈₈A₁₈₅.

It is evident that, under reducing conditions, human X and human Xa arein dimeric form; human Xa shows a lower molecular weight form of theheavy chain due to the absence of the activation peptide. Recombinanthuman X in lane 3 is similar to native human X, however some singlechain precursor is still evident. In lane 4, recombinant rX'Δ2 so showscleavage to the heavy and light chains. In lanes 5 and 6, the modifiedrecombinant Xi proteins behave in a manner similar to recombinant humanX. As expected, lanes 7 and 8 show the presence of monomer, heavy andlight chains derived from the proteolytic cleavage of X'i.

EXAMPLE 7 Enzymatic Analysis of Recombinant Human Factor X

The kinetic measurement of chromozym X(N-methoxycarbonyl-D-norleucyl-glycyl-arginine-4-nitranilide acetate,Boehringer Mannheim) hydrolysis by native human Factor X, Xa,recombinant X (rX), rX'Δ0, rX'Δ1, rX'Δ2, rX'Δ3, rXiN₈₈ A₁₈₅, rXiN₈₈,rX'i(Δ2)N₈₈ A₁₈₅ and inactivated bovine Xa, Xai-APMSF supplied by Dr. C.Esmon (OMRF, University of Oklahoma) (Skogen, W. F., et al., J Biol Chem(1984) 259:2306) were examined at room temperature in 96-well microtiterplates on a Molecular Devices Vmax spectrophotometer. The absorbance at405 nM was monitored continuously and the reaction velocities weredetermined directly by the machine and plotted with the Enzfitterprogram (Elsevier Press). Protein concentrations were determined byELISA (Example 6). All enzymes were diluted to the appropriateconcentrations in 0.1% bovine serum albumin (BSA) 50 mM Tris HCl, pH8.0, 150 mM NaCl. Duplicate reactions were carried out in 50 mM TrisHCl, pH 8.0, 150 mM NaCl and 2.5 mM CaCl₂. All recombinant human FactorX's were Mab717-purified (Example 6) except for RX'Δ0, rX'Δ1, and rX'Δ3which were purified using QAE-Sepharose (Pharmacia) concentrated(Skogen, W. F., et al., J Biol Chem (1984) 259:2306).

The recombinantly produced peptides derived from vectors containing rX,rXiN₈₈ A₁₈₅, rXiN₈₈ and rXaiA₁₈₅ were treated by preincubation for 5minutes with Russell's viper venom to convert them to the Xa or Xaiform. Peptides derived from the rX'Δ0, rX'Δ1, rX'Δ2 and rX'Δ3 vectorswere not treated in this fashion.

FIG. 7 is a comparison of Lineweaver-Burk plots for native human FactorX and Xa and activated forms derived from recombinant human rX and rX'.FIG. 7a, human X; FIG. 7b, human Xa; FIG. 7c, human rX (treated withRussell's viper venom protease); FIG. 7d, human rX' (not treated withprotease).

Table 2 compares the Kcat and Km values of the recombinantly producedhuman Factor X's to the native human Factor X and Xa supplied byHaematologic Technologies, Inc.

                  TABLE 2    ______________________________________                       Km     Specificity Constant               Kcat(s.sup.-1)                       (μm)                              Kcat/Km (s.sup.-1 M.sup.-1)    ______________________________________    Native forms    X            64        131    489 × 10.sup.3    Xa           367       184    1996 × 10.sup.3    Precursor construct    rX           22        134    167 × 10.sup.3    rX'Δ0  N.D.      -    rX'Δ1  N.D.      -    rX'Δ2  17        149    115 × 10.sup.3    rX'Δ3  N.D.    rXiN.sub.88 A.sub.185                 N.D.      -      -    rXiA.sub.185 N.D.      -      -    rX'i(Δ2)N.sub.88 A.sub.185                 N.D.      -      -    rX'i(Δ2)N.sub.88)2                 N.D.      -      -    Control CHO medium                 N.D.      -      -    ______________________________________     N.D. = not detected, Kcat ≦ .1 in 14 hrs - 16 hrs assay.

Of course, none of the inactivated forms give values; of the rX' forms,only rX'Δ2 showed activity.

EXAMPLE 8 Factor X Dependent Prothrombinase Complex Activity of Human X,Xa and Recombinant Human rX and rX'

Factor X dependent prothrombinase complex activity was determined bymeasuring the rate of chromozyme TH(tosyl-glycyl-prolyl-arginine-4-nitroanilide acetate, BoehringerMannheim) hydrolysis by thrombin at room temperature in a 96-wellmicrotiter plate on a Molecular Devices Vmax spectrophotometer. The.absorbance at 405 nM was continuously monitored and the initial oneminute reaction velocities were determined directly by the machine andplotted using the Enzfitter program (Elsevier). Reaction mixtures wereperformed in triplicate with 0.05×10⁻⁴ M to 1.5×10⁻⁹ M "Factor X,"determined by ELISA (Example 6), 0.5×10⁻⁶ M human prothrombin (STAGO,American Diagnostics, Inc.) 7.5×10⁻⁹ M human factor Va (HaematologicTechnologies, Inc.), 20×10-6M phosphocholine/phosphoserine 75%/25%(PCPS) (supplied by Dr. W. R. Church, University of Vermont), orequivalent amounts of rabbit brain cephalin (Sigma) (Example 9), 0.1%BSA (Sigma), 0.1×10⁻³ M chromozym TH (Boehringer Mannheim), 25 mM TrisHCl, pH 7.5, 150 mM NaCl and 5 mM CaCl₂.

Human Factor rX and rX' dependent prothrombinase complex activityutilized PCPS and human Factor X and Xa dependent prothrombinase complexactivity utilized cephalin. Human Factor X and rX were preincubated for5 minutes with Russell's viper venom (Haematologic Technologies, Inc.).Thrombin hydrolysis of chromozym TH as determined by increase offluorescence signal, was linear throughout the experimental protocol. Noobservable rates were shown for rXiN₈₈ A₁₈₅ at 59.2×10⁻⁴ M rX'iN₈₈ A₁₈₅at 10.2×10⁻⁹ M, or for bXai-APMSF at 1×10⁻⁹ M. FIGS. 8a-8d compareFactor X dependent prothrombinase complex activity of human X (FIG. 8a),human Xa (FIG. 8b) (Haematologic Technologies, Inc.), recombinant humanrX (after treatment with protease) (FIG. 8c) and recombinant human rX'Δ2(after no protease treatment) (FIG. 8d). All are comparably active.

EXAMPLE 9 Coagulation of Plasma

Mab717 purified rX and rXa were assayed for plasma coagulation activityin an automated two-stage prothrombin assay on a MLA Electra 800fibrometer. Enzyme protein concentrations were determined by ELISA(Example 6) and diluted in 0.1% BSA, 150 mM NaCl prior to use. BovineFactor X and Factor VII deficient plasma (Sigma) and rabbit braincephalin (Sigma) were prepared according to manufacturers' instructions.Russell's viper venom 0.1 μg/ml was added to human X and rX assays. Thereaction mixture comprised 0.1 ml Factor X, 0.1 ml 150 mM NaCl, 0.1 mlcephalin and 0.1 ml 25 mM CaCl₂. Duplicates were performed on eachconcentration and the average of two experiments were calculated. FIG. 9compares the plasma coagulation activity of human X, human Xa, human rXand human rXa. Human rX was calculated to be 45% as active as human Xand human rXa was calculated to be 32% as active as human Xa.

EXAMPLE 10 Inhibition of Prothrombinase. Complex Activity by rXiN₈₈A₁₈₅, Human rX'i(Δ2)N₈₈ A₁₈₅ and Bovine bXai-APMSF

Inhibition of native human Factor X dependent prothrombinase complexactivity by human rXiN₈₈ A₁₈₅ and inhibition of native human Factor5×10⁻⁹ M Xa dependent prothrombinase complex activity by humanrX'i(Δ2)N₈₈ A₁₈₅ (rXai) and bovine bXai-APMSF (C. Esmon, OMRF,University of Oklahoma) was tested as detailed in Example 8. It isnecessary to compare directly X with Xi and Xa with Xai because ofkinetic factors and the strength of the complex once formed. HumanrXiN₈₈ A815 was preincubated for 5 minutes with 0.1 μg/ml Russell'sviper venom. The human Factor X and Xa concentrations were 1×10⁻⁹ M.

FIG. 10 shows the concentration dependent inhibition of the human FactorXa dependent prothrombinase complex by bXai-APMSF, rX'i(Δ2)N₈₈ A₁₈₅ andinhibition of the human Factor X dependent prothrombinase complex byrXiN₈₈ A₁₈₅. 50% inhibition by bXai-APMSF was obtained at 0.9×10⁻⁹ M,50% inhibition by rX'i(Δ2)N₈₈ A₁₈₅ was obtained at 6×10⁻⁹ M and 50%inhibition by rXiN₈₈ A₁₈₅ was obtained at 10.6×10⁻⁹ M.

I claim:
 1. A single chain precursor polypeptide comprising the lightchain and heavy chain of Factor X in which at least a portion of thenative activation peptide sequence has been deleted and a proteolyticcleavage site has been inserted between the C-terminus of said lightchain and the N-terminus of said heavy chain, said polypeptide beingconvertible to Factor Xai by proteolysis and capable of competing withnative Factor Xa in the formation of a prothrombinase complex, and saidlight chain or said heavy chain being modified from its native aminoacid sequence so that said Factor Xai lacks protease activity whenincorporated into said prothrombinase complex.
 2. A precursorpolypeptide which is convertible to the Factor Xai of claim 1 byproteolysis.
 3. The polypeptide precursor of claim 1 which is reactivewith antibodies raised against Factor X.
 4. The single chain precursorpolypeptide of claim 1, in which the entire native activation peptidesequence has been deleted and said inserted proteolytic cleavage site islinked to the C-terminus of said light chain and the N-terminus of saidheavy chain.
 5. The single chain precursor polypeptide of claim 4, inwhich said proteolytic cleavage site has the sequence RKRRKR.
 6. Aprecursor polypeptide which is convertible to the Factor Xai of claim 4by proteolysis.
 7. The polypeptide precursor of claim 4 which isreactive with antibodies raised against Factor X.
 8. The single chainprecursor polypeptide of claim 1, wherein one or more of the residues atposition 42, 88 or 185 of said heavy chain are substituted by alternateamino acids.
 9. A method to prepare Factor Xai useful in treatment ofthrombosis, which method comprisescontacting the precursor polypeptideof claims 1, 4, 5 or 8 with an amount of a protease effective to cleavesaid precursor, and recovering the Factor Xai produced.
 10. The methodof claim 9 wherein said protease is contained in a Russell's viper venomextract.
 11. A two chain Factor Xai peptide modified from the nativeamino acid sequence of light chain positions 1-139 and heavy chainpositions 1-254 of FIG. 1, wherein said Factor Xai peptide is capable ofcompeting with Factor Xa in the formation of a prothrombinase complexand wherein said Factor Xai peptide does not result in proteolyticactivity when included in said complex, wherein a serine residue at aposition corresponding to that of position 185 of the heavy chain shownin FIG. 1 is replaced by an alternate amino acid and/or an aspartic acidresidue at a position corresponding to that of position 88 of the heavychain shown in FIG. 1 is replaced by an alternate amino acid and/or ahistidine residue at a position corresponding to that of position 42 ofthe heavy chain shown in FIG. 1 is replaced by an alternate amino acid.12. The Factor Xai of claim 11 wherein the replacement for serine may bean alanine or glycine and the replacement for aspartic acid may be anasparagine or glutamine and the replacement for histidine may be aphenylalanine residue.
 13. A single chain polypeptide which isconvertible to Factor Xai by proteolysis; said Factor Xai being amodified form of the amino acid sequence of light chain positions 1-139and heavy chain positions 1-254 of FIG. 1 wherein said Factor Xai iscapable of competing with Factor Xa in the formation of a prothrombinasecomplex and wherein said Factor Xai does not result in proteolyticactivity when included in said complex, wherein a serine residue at aposition corresponding to that of position 185 of the heavy chain shownin FIG. 1 is replaced by an alternate amino acid and/or an aspartic acidresidue at a position corresponding to that of position 88 of the heavychain shown in FIG. 1 is replaced by an alternate amino acid and/or ahistidine residue at a position corresponding to that of position 42 ofthe heavy chain shown in FIG. 1 is replaced by an alternate amino acid.14. The single chain polypeptide of claim 13 wherein the replacement forserine may be an alanine or glycine residue and the replacement foraspartic acid may be an asparagine or glutamine and the replacement forhistidine may be a phenylalanine residue.